4.HiSeq2000
企业: Illumina
推出时间: 2010/1/1
主流型号: Illumina HiSeq2000
样品要求: 6ug/sample,无RNA、蛋白污染，无降解，OD260/OD280=1.8-2.0
测序原理: 边合成边测序
这种测序技术通过将基因组DNA的随机片断附着到光学透明的表面，这些DNA片断通过延长和桥梁扩增，形成了具有数以亿计cluster的Flowcell，每个cluster具有约1000拷贝的相同DNA模板，然后用4种末端被封闭的不同荧光标记的碱基进行边合成边测序。这种新方法确保了高精确度和真实的一个碱基接一个碱基的测序，排除了序列方面的特殊错误，能够测序同聚物和重复序列。This technology attach random fragment of genome DNA to optically transparent surface.And by extention bridge amplification of the DNA fragments,it create a flow cell with > 10 million clusters, each containing ~1,000 copies of same template. Then sequence by synthese using four kind of terminal-closed base with different fluorescent mark. This technology assures the high accuracy and one by one base sequencing, avoids special mistakes of sequence and can be applied for repeated sequence and homopolymer.
文库制备: Fragment, Mate-pair (200bp-40kb)
模板制备: 桥式扩增
读长: 50SE,91PE,101PE 等
测序通量: 25G/day
时间/run:
91/101PE, 8-10 days ;
50SE, 3 days;
150PE, 15 days;
总体来说，策略不同，时间也有差别
碱基精确度: Q30%≥80%；Q20%≥90%
变异检测能力(DNA重测序方面): SNP、Indel、SV、CNV
成本: $690,000 per machine , $6000 per human genome sequencing(30X)
数据格式: 测序结果：*.fq;比对：*。SOAP/*.BAM/*.SAM;变异：*.gff
分析软件: GenomeStudio或者第三方软件包；自主选择
优点: 通量大，测序方式灵活，分析软件多样化
缺点: 在成本上目前高于第三代测序，样本制备过程复杂，样本要求相对 较高。
经典案例: Initial genome sequencing and analysis of multiple myeloma.Chapman MA,et al.Nature. 2011 Mar 24;471(7339):467-72.
Cytoplasmic intron sequence-retaining transcripts can be dendritically targeted via ID element retrotransposons.Buckley PT,et al.Neuron. 2011 Mar 10;69(5):877-84.
Identification of fusion genes in breast cancer by paired-end RNA-sequencing.Edgren H,et al. Genome Biol. 2011 Jan 19;12(1):R6.
Genome-wide association mapping to candidate polymorphism resolution in the unsequenced barley genome.Cockram J,et al. Proc Natl Acad Sci U S A. 2010 Dec 14;107(50):21611-6. Epub 2010 Nov 29.
MHC class II transactivator CIITA is a recurrent gene fusion partner in lymphoid cancers.Steidl C, et al.Nature. 2011 Mar 17;471(7338):377-81. Epub 2011 Mar 2.
Tumour evolution inferred by single-cell sequencing.Navin N,et al.Nature. 2011 Apr 7;472(7341):90-4. Epub 2011 Mar 13.
备注: 是目前市场上最主流的测序仪器，demo case众多；另外，The additional benefit is experimental flexibility- applications that require different read lengths can be run on each flowcell – an example is that a whole genome sequencing and de novo sequencing study (eg. Of human and microorganism) could be run on one flowcell at 2 x 100bp reads, and the other could be running ChIP-seq and gene expression samples at 1 x 50bp read length. Each flowcell is controlled independently; hence, a run can be started/stopped independent of the other.
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